Chronic beryllium disease is a granulomatous lung disease associated with a pathologic cell-mediated immune response to inhaled particles of beryllium metal. It offers a unique opportunity to study the immunologic and inflammatory events that occur in the progression from antigen exposure to antigen sensitization to chronic inflammation and disease. The central focus of this NIH- and CDC/NIOSH-funded research project has been to examine the populations of antigen-specific T cells that emerge in the lungs of patients with disease, to study their interaction with lung macrophages resulting in initiation and amplification of the granulomatous response, and to track longitudinally the natural history of beryllium sensitization and disease in a population of affected individuals. The precise physiologic role of T lymphocytes in the development of granuloma is unknown. One of our major objectives has been to characterize the role of T lymphocytes both in the recognition of inhaled antigens and in the amplification of the immune response, leading to disease. Only a limited number of T cells in a granuloma are known to express receptors on their surface that can recognize inciting antigens. We have previously observed that within the lungs of patients with the granulomatous disease sarcoidosis, a limited set of subfamilies of T cells emerge, presumably in order to recognize the as yet unknown antigen causing that disease (Forrester 1993; Forrester 1994) Because the antigen is known in beryllium disease, and because of compartmentalization of the beryllium response to the lungs (Newman 1994), we were able to test how lung T cell exposure to a disease-causing antigen influences the accumulation of T cell subpopulations bearing particular T cell antigen receptors in vitro. Using two complementary methods--flow cytometric analysis with monoclonal antibodies directed against portions of the T cell receptor complex and DNA sequencing of key gene segments of the T cell antigen receptor--we have been able to demonstrate that there is a limited subset of T cells that can recognize the inhaled beryllium that is presented to the T cell by an antigen presenting cell (Fontenot 1997). This small cadre of T cell clones suggests that there may be a limited number of configurations that the antigen may assume when it is presented to T cells, and has led to our current work on sequencing the entire binding region of the human lung T cell antigen receptor in patients with chronic beryllium disease. In the past year we have successfully cloned beryllium-reactive T cells from the lungs of patients with CBD. We have sequenced their VB3 and matching Valpha T cell receptor partners and demonstrated their clonality as well as their persistence within individuals over time and among individuals who showed high VB3-bearing T cell numbers in their lavage compared to blood. While there would not be granulomas without antigen recognition and sensitization, T cell recognition of beryllium is probably not sufficient to produce disease. We hypothesize that immune effector cells produce several key cytokines that lead to the pathologic amplification of the cellular immune response. We have observed that beryllium-stimulated lymphocytes from patients with beryllium disease produce two key factors-- IL-2 and gamma-interferon--that together help drive the amplification of the cell-mediated immune response to antigen (Tinkle 1996; Tinkle 1997). These lung lavage cells do not secrete IL-4 and show no increase in IL-4 specific-mRNA based on RT-PCR analysis, thus confirming that they are preferentially of the so-called "T helper 1" (Th1) phenotype, favoring cell-mediated immune responses. When beryllium-reactive T cells are stimulated with beryllium salts in vitro, gamma-interferon protein and mRNA levels are unusually elevated and unusually sustained, suggesting that beryllium itself or some secondarily stimulated cytokine is regulating gamma-interferon production at the transcriptional level (Tinkle 1997). We have also shown that the excessive T cell proliferation and gamma-interferon production in beryllium disease observation cannot be explained by a failure of IL-10 downregulation (Tinkle 1996). In the last year we have begun to test the hypothesis that beryllium, which is known to bind nuclear proteins, may be directly turning on gamma-interferon by influencing nuclear factor binding to the gamma interferon gene promoter. As a first step toward that goal, we first established whether activation of class II MHC (HLA-DR or -DP) on the surface of beryllium reactive T cells is associated with the gamma interferon upregulation. Anti-MHC antibody binding did not stop beryllium-induced gamma interferon production. Secondly, we tested whether the relative absence of IL-4 explained the unchecked production of gamma interferon. Addition of IL-4 did not change gamma interferon production. In the clinical research vein, we continue to track two populations of beryllium-affected individuals: those who are sensitized but who do not yet have disease, and those who have granulomatous lung disease but who are at a subclinical stage. It is not known if patients who are beryllium sensitized will all develop disease or if patients who are found to have subclinical granulomatous pathology in their lungs will necessarily progress to clinical illness. These natural history studies have tracked these populations of patients for an average of 4 years, with serial examinations, pulmonary function tests, chest imaging, and lung lavage. Thus far we can preliminarily conclude that beryllium sensitization leads to beryllium disease in at least a subset of individuals; similarly, a subset of individuals who have early granulomatous lung involvement develop clinically-significant illness. Over time we expect to be able to calculate the frequency and the rate of progression and hope to identify risk factors for disease progression that will allow us to better prognosticate and to determine who should receive treatment. Preliminary data suggest that beryllium sensitization progresses into chronic beryllium disease at a rate of approximately 10% per year. It is not yet known whether all sensitized individuals will eventually develop disease, but it is clear that a significant proportion of people detected as having sensitization are at increased disease risk.